Transplantation of cells into the nasal cavity and the subarachnoid cranial space

ABSTRACT

A method of transplanting cells into a subject is disclosed. The method comprises transplanting the cells into the paranasal sinus of the subject or the subarachnoid cavity situated between the frontal bone of skull and the olfactory bulb of the subject. Devices for paranasal sinus transplantation and subarachnoid cavity transplantation are also disclosed.

RELATED APPLICATIONS

This application is a continuation application of U.S. patentapplication Ser. No. 13/916,743 filed on Jun. 13, 2013 which claims thebenefit of priority under 35 USC 119(e) of U.S. Provisional PatentApplication Nos. 61/659,456 filed Jun. 14, 2012 and 61/773,263 filedMar. 6, 2013. The contents of all of the above applications areincorporated by reference as if fully set forth herein.

FIELD AND BACKGROUND OF THE INVENTION

The present invention, in some embodiments thereof, relates to a methodof transplanting cells into the nasal cavity and, more particularly, butnot exclusively, into the paranasal sinuses (PNS), and into thesubarachnoid cavity.

Cell therapy is one of the most promising future techniques in themedical arsenal for the repair of damaged or destroyed tissue and/or forthe replacement of dysfunctional cells. The diseases which cell therapycan target are very varied: Hormonal dysfunction, such as diabetes andgrowth hormone deficiency; neurodegenerative diseases, such asParkinson's, Alzheimer's and Huntington's; and cardiovascular lesions,such as myocardial infarction, peripheral vascular ischaemia; as well aslesions in the cornea, skeletal muscle, skin, joints and bones etc. Theobjective of cell therapy is to restore the lost function rather thanproduce a new organ, which could cause duplicity and undesirableeffects. Several resources of cells can be used to restore the damagedtissue, such as donor cells from human cadavers, resident stem cells,multipotent adult progenitor cells or embryonic stem cells.

Type 1 Diabetes Mellitus (T1DM) as well as Type 2 (T2DM) are bothdiseases resulting from pancreatic islets dysfunction and insulindependency. Islet transplantation is a potential successful treatmentfor T1DM patients and a subgroup of patients with T2DM. Currently,clinical trials in humans such as the “Edmonton protocol” show thatlong-term outcomes of islet transplantation into the liver are hamperedby a gradual decrease in islet function occurring during the early posttransplantation period. The dramatic initial loss of islets is believedto be related to hypoxia at the transplantation site. In addition, theliver site requires an immunosuppressive therapy and is associated withprocedure-related complications, including haemorrhage and thrombosis.The spleen, eye, brain, thymus, testes, pancreas, kidney capsule,peritoneum, bone morrow, lung, subcutis, muscle and omental pouch havebeen explored as potential sites for islet transplantation. Currently,experimental islet transplantations in these sites are hampered byinsufficient oxygenation, immune rejection, limited space andcomplicated surgical procedure (Shapiro A J et al, Curr Diab Rep. 2011;11(5):345-54; Merani et al., Br J Surg. 2008; 95(12):1449-61).

Firouzi M et al., Neurosci Lett. 2006 Jul. 10; 402(1-2):66-70, teachestransplantation of Schwann cells into the subarachnoid cavity.

SUMMARY OF THE INVENTION

According to an aspect of some embodiments of the present inventionthere is provided a method of transplanting cells into a subject in needthereof comprising transplanting the cells into the paranasal sinus ofthe subject or the subarachnoid cavity situated between the frontal boneof the skull and the olfactory bulb of the subject, therebytransplanting the cells.

According to an aspect of some embodiments of the present inventionthere is provided a method of treating a disease in a subject in needthereof, comprising transplanting a therapeutically effective amount ofcells into the paranasal sinus of the subject or the subarachnoid cavitysituated between the frontal bone of the skull and the olfactory bulb ofthe subject, thereby treating the disease.

According to an aspect of some embodiments of the present inventionthere is provided a method of treating a disease in a subject in needthereof, comprising administering a therapeutically effective amount ofcell aggregates into the nasal cavity of the subject or the subarachnoidcavity situated between the frontal bone of skull and the olfactory bulbof the subject, wherein said cell aggregates are greater than 50 micronsin diameter, thereby treating the disease.

According to an aspect of some embodiments of the present inventionthere is provided an endoscopic device comprising a container attachedto a flexible tubing and an optic fiber, wherein the inner surface ofthe container is coated with an agent that prevents adhesiveness of cellaggregates thereto.

According to some embodiments of the invention, when the disease isDiabetes, the cell aggregates comprises islets.

According to some embodiments of the invention, the disease is anautoimmune disease.

According to some embodiments of the invention, the disease is selectedfrom the group consisting of a hormone deficiency disease, a clottingfactor disorder, a brain disorder and an enzyme deficiency disease.

According to some embodiments of the invention, the autoimmune diseaseis selected from the group consisting of Diabetes, Hashimotos throiditisand Addison disease.

According to some embodiments of the invention, the hormone of thehormone deficiency disease is selected from the group consisting ofinsulin, thyroxine, growth hormone, testosterone, oestrogen,erythropoietin and aldosterone.

According to some embodiments of the invention, the enzyme of the enzymedeficiency disease is selected from the group consisting of lysosomalenzyme is selected from the group consisting of glucocerebrosidase(GCD), acid sphingomyelinase, hexosaminidase,α-N-acetylgalactosaminidise, acid lipase, α-galactosidase,α-L-iduronidase, iduronate sulfatase, α-mannosidase, sialidase, αfucosidase, G_(M1)-β-galctosidase, ceramide lactosidase, arylsulfataseA, β galactosidase and ceramidase.

According to some embodiments of the invention, the clotting factordisease is hemophilia A.

According to some embodiments of the invention, the brain disorder is aneurodegenerative disorder.

According to some embodiments of the invention, the cells comprisegenetically modified cells.

According to some embodiments of the invention, the cells express arecombinant polypeptide.

According to some embodiments of the invention, the cells are seeded ona scaffold prior to the transplanting.

According to some embodiments of the invention, the method furthercomprises inserting a scaffold into the paranasal sinus or thesubarachnoid cavity prior to the transplanting.

According to some embodiments of the invention, the cells comprise stemcells.

According to some embodiments of the invention, the cells compriseimmunoisolated cells.

According to some embodiments of the invention, the cells comprisepancreatic beta cells.

According to some embodiments of the invention, the cells are autologousto the subject.

According to some embodiments of the invention, the cells arenon-autologous to the subject.

According to some embodiments of the invention, the paranasal sinusesare selected from the group consisting of the maxillary sinuses, frontalsinuses, ethmoid sinuses, and the sphenoid sinuses.

According to some embodiments of the invention, the method furthercomprises administering to the subject an immunosuppressive agent.

According to some embodiments of the invention, the transplanting iseffected by endoscopy.

According to some embodiments of the invention, the cell aggregatescomprise pancreatic islets.

According to some embodiments of the invention, the inner diameter ofthe tubing is between about 200 μm-1000 μm.

According to some embodiments of the invention, the container comprisesa population of cells.

Unless otherwise defined, all technical and/or scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which the invention pertains. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of embodiments of the invention, exemplarymethods and/or materials are described below. In case of conflict, thepatent specification, including definitions, will control. In addition,the materials, methods, and examples are illustrative only and are notintended to be necessarily limiting.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawings will be provided by the Office upon request and paymentof the necessary fee.

Some embodiments of the invention are herein described, by way ofexample only, with reference to the accompanying drawings. With specificreference now to the drawings in detail, it is stressed that theparticulars shown are by way of example and for purposes of illustrativediscussion of embodiments of the invention. In this regard, thedescription taken with the drawings makes apparent to those skilled inthe art how embodiments of the invention may be practiced.

In the drawings:

FIGS. 1A-1C are a pictorial illustration of the position of theparanasal sinuses and nasal path for non-invasive cell delivery intodifferent sinuses.

FIG. 2 is a graph illustrating the hypoglycemic effect of insulinomaINS-1 cell transplanted into the paranasal sinus (PNS) of streptozotocin(STZ)-diabetic rats. Diabetes was induced in 5 male Lewis rats by i.v.injection of 85 mg STZ/kg. Rats were pretreated with 10 mg/kg CyA 2 daysprior to transplantation followed by a daily dosage of CyA. One weekafter STZ injection, about 30×10⁶ cells were implanted (Tx) in PNS ofdiabetic rats. Head was removed for immunohistochemical analysis twoweeks following cell implantation.

FIGS. 3A-3B are photographs photograph of a PNS implantation site underlow (FIG. 3A) and high (FIG. 3B) magnifications Immunoperoxidase insulinstaining (brown color) of rat insulinoma cells INS-1 implanted in PNS.

FIG. 4 is a diagram of an exemplary device which can be used totransplant the cells into the nasal and/or paranasal cavity of asubject.

FIGS. 5A-5D illustrate long-term glucose metabolism after islettransplantation in subarachnoid cavity of diabetic rats. A. Non fastingblood glucose profile. Glucose concentrations in a group of nontransplanted STZ-diabetic animals were more than 350 mg/dl for theexperimental period. B. Intraperitoneal test tolerance to glucose(IPTTG). IPTTG was performed by injecting glucose at a concentration of1 g/kg body weight following 6 h fast. Blood glucose was monitored bysampling from the tail vein periodically up to 120 min as indicated. Thedata represent the mean±SD of five independent rats in a group of intactanimals (Intact) and in a group of non transplanted STZ-diabetic animals(STZ) and three rats in a group of transplanted STZ-diabetic animals(STZ+transplant). Arrows show the time of STZ-treatment and islettransplantation (Tr). C Immunohistochemical image of islets transplantedin the subarachnoid cavity of rats treated with STZ: peroxidase stainingof insulin (brown). D Immunofluorescent double staining of isletstransplanted in the subarachnoid cavity of rats treated with STZ(insulin in red, glucagon in green). Tissues for histological analysiswere taken two months after transplantation. Scale bars −200 μm.

FIG. 6 Schematic presentation of possible anti-diabetic andanti-dementia effects of islets transplanted in subarachnoid cavity. A.The arrows show pathways for the oxygen, glucose and nutrition supply tothe transplanted islets from surrounding tissues and the CSF, as well ashormone delivery and metabolic waste removal from grafted islets. B. Therole of subarachnoid cavity/CSF in facilitating islet hormone transportinto the blood and direct hormone delivery into the brain. C. Effects onglucose homeostasis and cognitive functions.

FIGS. 7A-7B Burr hole position in rat skull. A. Schematic presentation(black circle indicates position of burr hole in the frontal bone of theskull). B. Microscopy image of a rat skull with position of burr hole(bold arrow) and transplanted islets stained for insulin. Scale bar−2000 μm.

DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

The present invention, in some embodiments thereof, relates to a methodof transplanting cells and, more particularly, but not exclusively, intothe paranasal sinuses or the subarachnoid cavity situated between thefrontal bone of the skull and the olfactory bulb of the subject.

Before explaining at least one embodiment of the invention in detail, itis to be understood that the invention is not necessarily limited in itsapplication to the details set forth in the following description orexemplified by the Examples. The invention is capable of otherembodiments or of being practiced or carried out in various ways.

Various sites have been explored for the transplantation of pancreaticislets including the spleen, eye, brain, thymus, testes, pancreas,kidney capsule, peritoneum, bone morrow, lung, subcutis, muscle andomental pouch. Currently, experimental islet transplantations in thesesites are hampered by insufficient oxygenation, immune rejection,limited space and complicated surgical procedures.

The present inventors propose two new sites for transplantation, namelythe highly oxygenated air-filled paranasal sinuses and the subarachnoidcavity situated between the frontal bone of the skull and the olfactorybulb.

Human paranasal sinuses are composed of a group of four pairedair-filled spaces surrounding the nasal cavity (maxillary sinuses),above the eyes (frontal sinuses), between the eyes (ethmoid sinuses),and behind the ethmoids (sphenoid sinuses).

When compared to the currently tested alternative sites for islettransplantations, the paranasal sinuses have important advantages:

1. Easy access, allowing for a noninvasive or minimally invasiveprocedure of cell implantation in humans;

2. Excellent physiological oxygenation due to continuous aeration duringrespiration (pO2=96 mmHg in PNS v.s pO2=5 mmHg in islets transplantedinto liver; and

3. Sufficient space in the case of islet transplantation (human PNSvolume is ˜60-80 cm³; vs. transplanted islets volume of ˜1-2 cm³).

Whilst reducing the present invention to practice, the present inventorshave shown that STZ-diabetic rats became hypoglycemic during one weekfollowing transplantation of insulin producing INS cells into the PNS(FIG. 2).

The olfactory bulb is known to be enriched with insulin receptor,displays the highest transport rate for insulin in the brain and playsan important role in development of different neurodegenerativediseases, particularly in Alzheimer's and Parkinson's diseases. It hasbeen shown that decreased brain insulin levels and/or signaling areassociated with impaired learning, memory and various neurodegenerativediseases.

Whilst further reducing the present invention to practice, the presentinventors showed that islet cell transplantation at a particularposition in the subarachnoid cavity (i.e. situated between the frontalbone of the skull and the olfactory bulb) enabled the assembly ofgrafted islets directly onto the glomeruli of the olfactory bulb. Asillustrated in FIGS. 5A-D, severely diabetic rats which weretransplanted with islets at this site became normoglycemic after twodays and demonstrated normal glucose tolerance typical for healthyanimals,

Thus, according to one aspect of the present invention, there isprovided a method of transplanting cells into a subject in need thereofcomprising transplanting the cells into the nasal cavity of the subjector the subarachnoid cavity situated between the frontal bone of theskull and the olfactory bulb, thereby transplanting the cells.

Nasal Cavity:

The phrase “nasal cavity” refers to air filled space above and behindthe nose in the middle of the face including the paranasal sinuses.According to one embodiment, the administration is effected through thenose (e.g. via the nostrils). According to another embodiment, theadministration is effected via the mouth.

According to one embodiment, the transplanting is into the paranasalsinus.

According to this aspect of the present invention, the term“transplanting into the paranasal sinus” refers to administration intoat least one paranasal sinus of the subject. Preferably, at least about20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% of the transplanted cells reachthe paranasal sinus at least 5 minutes following transplantation.

Preferably, at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% of thetransplanted cells reach the paranasal sinus at least 1 minute followingtransplantation.

Preferably, at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% of thetransplanted cells reach the paranasal sinus at least 20 secondsfollowing transplantation.

According to one embodiment, the transplanting is local—i.e. the cellsenter the cavity directly by the act of transplantation and not bysystemic routes, and do not migrate from a different transplantationsite outside the paranasal sinus. It will be appreciated that followingthe transplantation, the cells are capable of migrating out of thetransplantation site (paranasal sinus) to act systemically.

According to one embodiment, the transplanting is effected byintroduction of the cellular composition into the body using a device(e.g. via endoscopy), wherein at least a portion of the device entersthe nose (and more preferably the paranasal cavity of the sinus).Further description of the device is provided herein below.

The paranasal sinuses may be any one of the maxillary sinuses, frontalsinuses, ethmoid sinuses, and the sphenoid sinuses as illustrated inFIGS. 1A-C.

Subarachnoid Cavity:

As used herein, the term “subarachnoid cavity” refers to the space inthe meninges beneath the arachnoid membrane and above the pia mater thatcontains the cerebrospinal fluid.

In order to transplant, into the subarachnoid cavity, preferably thefrontal bone is pierced and the dura mater and the arachnoid membraneperforated.

The position of insertion of the transplant should be located such thatthe cells are capable of diffusing onto the olfactory bulb.

Preferably, at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% ofthe transplanted cells reach the olfactory bulb at least 5 minutesfollowing transplantation.

Preferably, at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% ofthe transplanted cells reach the olfactory bulb at least 1 minutefollowing transplantation.

Preferably, at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% ofthe transplanted cells reach the olfactory bulb at least 20 secondsfollowing transplantation.

According to one embodiment, the transplanting is local—i.e. the cellsenter the subarachnoid cavity directly by the act of transplantation andnot by systemic routes, and do not migrate from a differenttransplantation site outside the subarachnoid cavity. It will beappreciated that following the transplantation, the cells are capable ofmigrating out of the transplantation site (subarachnoid cavity) to actsystemically.

The composition is a “transplant”, and the mammal is the recipient. Thetransplant and recipient may be syngeneic, allogenic, or xenogeneic.

The term “cells” as used herein, refers to embryonic, fetal, pediatric,or adult cells or tissues, including but not limited to, stem cells,induced pluripotent stem cells, precursors cells, and progenitor cells.

The cell may be in an isolated form (e.g. in a single cell suspension),may be part of a cell aggregate (e.g. comprised in a pancreatic islet)or may be part of a tissue or organ.

As used herein, the term “cell aggregate” refers to a plurality of cellsthat are connected by a physical interaction—i.e. a cell cluster. Thecell aggregate may comprise 20 cells, 100 cells, 500 cells, 1000 cells,5000 cells or more. The cell aggregate may comprise more than one typeof cell. Thus, for example, an islet comprises alpha cells producingglucagon (15-20% of total islet cells), beta cells producing insulin andamylin (65-80%), delta cells producing somatostatin (3-10%), PP cellsproducing pancreatic polypeptide (3-5%) and epsilon cells producingghrelin (<1%).

According to one embodiment, the cell aggregate is between about 50μm-500 μm, 100 μm-500 μm, 100 μm-300 μm or 200-400 μm in diameter.

The cells may be derived from a primary culture or may be derived from acell line.

The cells may be fresh, frozen or preserved in any other way known inthe art (e.g. cryopreserved).

According to another embodiment, the cells are derived from the pancreasor the liver.

Typically, the cells secrete a factor (e.g. a polypeptide) that isuseful for the treatment of a disease.

Such factors include for example, hormones including but not limited toinsulin, thyroxine, growth hormone, testosterone, oestrogen,erythropoietin and aldosterone; enzymes, including but not limited tolysosomal enzyme such as glucocerebrosidase (GCD), acidsphingomyelinase, hexosaminidase, α-N-acetylgalactosaminidise, acidlipase, α-galactosidase, α-L-iduronidase, iduronate sulfatase,α-mannosidase, sialidase, a fucosidase, G_(M1)-β-galctosidase, ceramidelactosidase, arylsulfatase A, β galactosidase and ceramidase; clottingfactors such as factor VIII.

According to a preferred embodiment, the cells secrete pancreatichormones (e.g. insulin, glucagon, somatostatin).

As used herein, the term “pancreatic hormones” refers to hormonesobtained by synthesis or recombination, in which the peptide sequence isthe sequence of human hormone, includes the allelic variations and thehomologs. The polypeptide sequence of the hormone may be modified toimprove the function of the hormone (e.g. long lasting).

Cells which secrete neurotrophic factors are also contemplated by thepresent invention.

As used herein, the phrase “neurotrophic factor” refers to a cell factorthat acts on the cerebral nervous system comprising growth,differentiation, functional maintenance and/or survival effects onneurons. Examples of neurotrophic factors include, but are not limitedto, glial derived neurotrophic factor (GDNF), GenBank accession nos.L19063, L15306; brain-derived neurotrophic factor (BDNF), GenBankaccession no CAA62632; neurotrophin-3 (NT-3), GenBank Accession No.M37763; neurotrophin-4/5; Neurturin (NTN), GenBank Accession No.NP_004549; Neurotrophin-4, GenBank Accession No. M86528; Persephin,GenBank accession no. AAC39640; brain derived neurotrophic factor,(BDNF), GenBank accession no. CAA42761; artemin (ART), GenBank accessionno. AAD13110; ciliary neurotrophic factor (CNTF), GenBank accession no.NP_000605; insulin growth factor-I (IGF-1), GenBank accession no.NP_000609; and Neublastin GenBank accession no. AAD21075.

Cells which secrete neuropeptides are also contemplated by the presentinvention. Examples of neuropeptides include, but are not limited toOxytocin, Vasopressin, Corticotropin releasing hormone (CRH), Growthhormone releasing hormone (GHRH), Luteinizing hormone releasing hormone(LHRH), Somatostatin growth hormone release inhibiting hormone,Thyrotropin releasing hormone (TRH), Neurokinin α (substance K),Neurokinin β, Neuropeptide K, Substance P, β-endorphin, Dynorphin, Met-and leu-enkephalin, Neuropeptide tyrosine (NPY), Pancreatic polypeptide,Peptide tyrosine-tyrosine (PYY), Glucogen-like peptide-1 (GLP-1),Peptide histidine isoleucine (PHI), Pituitary adenylate cyclaseactivating peptide (PACAP), Vasoactive intestinal polypeptide (VIP),Brain natriuretic peptide, Calcitonin gene-related peptide (CGRP) (α-and β-form), Cholecystokinin (CCK), Galanin, Islet amyloid polypeptide(IAPP), Melanin concentrating hormone (MCH), ACTH, α-MSH, NeuropeptideFF, Neurotensin, Parathyroid hormone related protein, Agoutigene-related protein (AGRP), Cocaine and amphetamine regulatedtranscript (CART)/peptide, Endomorphin-1 and -2, 5-HT-moduline,Hypocretins/orexins Nociceptin/orphanin FQ, Nocistatin, Prolactinreleasing peptide, Secretoneurin and Urocortin.

Cells which secrete neurotransmitters are also contemplated by thepresent invention.

A neurotransmitter according to the teaching of the present inventioncan be any substances which is released on excitation from the axonterminal of a presynaptic neuron of the central or peripheral nervoussystem and travel across the synaptic cleft to either excite or inhibitthe target cell. The neurotransmitter can be, for example, dopamine,norepinephrine, epinephrine, gamma aminobutyric acid, serotonin,acetylcholine, glycine, histamine, vasopressin, oxytocin, a tachykinin,cholecytokinin (CCK), neuropeptide Y (NPY), neurotensin, somatostatin,an opioid peptide, a purine or glutamic acid.

Examples of diseases that may be treated using the cells describedherein include, but are not limited to an autoimmune disease (e.g.Diabetes type I or type II), Hashimotos throiditis and Addison disease,a clotting factor disorder (e.g. Hemophilia), a brain disorder (e.g.neurodegenerative disorders such as Alzheimer's or Parkinson's disease),a disease or disorder associated with under-production of a hormone andan enzyme deficiency disease. Since the cells of the present inventionmay be selected to secrete a particular hormone, it is contemplated thatthe cells may also be useful for the treatment of reproductivedisorders, contraception or to increase fertility.

Table 1 below provides a list of brain diseases together with theimportant factor useful for treating the diseases.

TABLE 1 REF factors Disease Walker D G, et al. Brain Res 1998; 794:181-7. BDNF, FGF, Parkinson's Lorigados L, et al. Rev Neurol 1998; 26:744-8. GDNF Mogi M, et al. Neurosci Lett 1994; 180: 147-50. Howells D W,et al. Exp Neurol 2000; 166: 127-35. Beck K D, et al. Nature 1995; 373:339-41. Tomac A, et al. Nature 1995; 373: 335-9. Gash D M, et al. Nature1996; 380: 252-5. Choi-Lundberg D L, Science 1997; 275: 838-41. Bozzi Y,Borrelli E. Eur J Neurosci 1999; 11: 1275-84. Chauhan N B, et al SocNeurosci Abstr 1998; 24: 1465. Chauhan N B, et al, Neurology 1999; 52:A212-213. G. W. Mathern, Mol. Chem. Neuropathol. 30 1-2 (1997), BDNF,NT-3, Epilepsy pp. 53-76. Lucia Tapia-Arancibia et al. Frontiers inNeuroendocrinology 2004 July; 25(2): 77-107. RYUTA KOYAMA and YUJIIKEGAYA; NEUROSCIENCE UPDATE 2005 August; 11(4): 282-7. Gerald Seifert,et al., Nature Reviews Neuroscience 7, 194-206 (March 2006). Luis H. Etal., Brain Research Reviews 2004 December; NT3, IGF1, BDNF, ALS 47(1-3):263-74. Bradley W G. Ann Neurol 1995; 38: 971. Haase G, et al. Nat Med1997; 3: 429-36. Arakawa Y, J Neurosci 1990; 10: 3507-15. Ron D, Janak PH. Rev Neurosci. 2005; 16(4): 277-85. GDNF Drug and alcohol addictionCrutcher K A, et al. J Neurosci 1993; 6: 2540-50. BDNF Alzheimer's ScottS A, et al. Nerve growth factor in Alzheimer's disease: increased levelsthroughout the brain coupled with declines in nucleus basalis. JNeurosci 1995; 15: 6213-21. Peng S, et al. J Neuropathol Exp Neurol2004; 63: 641-9. Murer M G, et al. Neuroscience 1999; 88: 1015-32.Martinez-Serrano A, Bjorklund A. Trends Neurosci 1997; BDNF, NT-3,Huntington's 20: 530-8. or NT-4/5 Perez-Navarro E, et al. J Neurochem2000; 75: 2190-9. Perez-Navarro E, et al. Neuroscience 1999; 91:1257-64. Gal Shoval, Abraham Weizmana; Eur NT-3 BDNF SchizophreniaNeuropsychopharmacol. 2005 May; 15(3): 319-29. Levi-Montalcini, R.,1987. Biosci. Rep. 7, 681-699. Hattori, M., Nanko, S., 1995. Biochem.Biophys. Res. Commun. 209, 513-518. Virgos, C., 2001, Schizophr. Res.49, 65-71. Paul A. Sieving, et al., Proc Natl Acad Sci USA. 2006 CNTFOptic nerve Mar. 7; 103(10): 3896-901. Wu D; Neuro Rx. 2005 January;2(1): 120-8. FGF, BDNF Stroke

It will be appreciated that when the transplantation site is thesubarachnoid cavity, insulin producing cells (e.g. pancreatic isletcells) may be used to treat brain disorders such as dementia and otherneurodegenerative diseases (for example, those listed herein above)since it is known that adequate insulin supply to the brain restorescerebral glucose homeostasis and cognitive functions.

According to one embodiment, the cells are genetically modified toexpress a recombinant protein. Preferably, the recombinant protein issecreted from the cell.

Examples of recombinant proteins that may be expressed (and preferablysecreted) in the cells of the present invention include, but are notlimited to an antibody, insulin, human growth hormone (rHGH), folliclestimulating hormone, factor VIII, erythropoietin, Granulocytecolony-stimulating factor (G-CSF), alpha-glactosidase A,alpha-L-iduronidase (rhIDU; laronidase),N-acetylgalactosamine-4-sulfatase (rhASB; galsulfase) Tissue plasminogenactivator (TPA), Glucocerebrosidase, Interferon (IF) Interferon-beta-1a,Interferon beta-1b, Insulin-like growth factor 1 (IGF-1), somatotropin(ST) and chymosin.

The cells may also be genetically modified to express apolynucleotide—e.g. siRNA sequence or an miRNA sequence.

According to one embodiment, the polynucleotide sequence is a dsRNA.

The polynucleotide sequence may be incorporated into a nucleic acidconstruct, as further described herein below or may be introduced perse, (i.e. without incorporating into a construct).

To express exogenous a recombinant polypeptide, a polynucleotidesequence encoding the polypeptide is preferably ligated into a nucleicacid construct suitable for mammalian cell expression. Such a nucleicacid construct includes a promoter sequence for directing transcriptionof the polynucleotide sequence in the cell in a constitutive orinducible manner

Constitutive promoters suitable for use with some embodiments of theinvention are promoter sequences which are active under mostenvironmental conditions and most types of cells such as thecytomegalovirus (CMV) and Rous sarcoma virus (RSV). Inducible promoterssuitable for use with some embodiments of the invention include forexample the tetracycline-inducible promoter (Zabala M, et al., CancerRes. 2004, 64(8): 2799-804).

The nucleic acid construct (also referred to herein as an “expressionvector”) of some embodiments of the invention includes additionalsequences which render this vector suitable for replication andintegration in prokaryotes, eukaryotes, or preferably both (e.g.,shuttle vectors). In addition, a typical cloning vectors may alsocontain a transcription and translation initiation sequence,transcription and translation terminator and a polyadenylation signal.By way of example, such constructs will typically include a 5′ LTR, atRNA binding site, a packaging signal, an origin of second-strand DNAsynthesis, and a 3′ LTR or a portion thereof.

The nucleic acid construct of some embodiments of the inventiontypically includes a signal sequence for secretion of the peptide from ahost cell in which it is placed. Preferably the signal sequence for thispurpose is a mammalian signal sequence or the signal sequence of thepolypeptide variants of some embodiments of the invention.

Eukaryotic promoters typically contain two types of recognitionsequences, the TATA box and upstream promoter elements. The TATA box,located 25-30 base pairs upstream of the transcription initiation site,is thought to be involved in directing RNA polymerase to begin RNAsynthesis. The other upstream promoter elements determine the rate atwhich transcription is initiated.

Preferably, the promoter utilized by the nucleic acid construct of someembodiments of the invention is active in the specific cell populationtransformed. Examples of cell type-specific and/or tissue-specificpromoters include promoters such as albumin that is liver specific[Pinkert et al., (1987) Genes Dev. 1:268-277], lymphoid specificpromoters [Calame et al., (1988) Adv. Immunol. 43:235-275]; inparticular promoters of T-cell receptors [Winoto et al., (1989) EMBO J.8:729-733] and immunoglobulins; [Banerji et al. (1983) Cell 33729-740],neuron-specific promoters such as the neurofilament promoter [Byrne etal. (1989) Proc. Natl. Acad. Sci. USA 86:5473-5477], pancreas-specificpromoters [Edlunch et al. (1985) Science 230:912-916] or mammarygland-specific promoters such as the milk whey promoter (U.S. Pat. No.4,873,316 and European Application Publication No. 264,166).

Enhancer elements can stimulate transcription up to 1,000 fold fromlinked homologous or heterologous promoters. Enhancers are active whenplaced downstream or upstream from the transcription initiation site.Many enhancer elements derived from viruses have a broad host range andare active in a variety of tissues. For example, the SV40 early geneenhancer is suitable for many cell types. Other enhancer/promotercombinations that are suitable for some embodiments of the inventioninclude those derived from polyoma virus, human or murinecytomegalovirus (CMV), the long term repeat from various retrovirusessuch as murine leukemia virus, murine or Rous sarcoma virus and HIV.See, Enhancers and Eukaryotic Expression, Cold Spring Harbor Press, ColdSpring Harbor, N. Y. 1983, which is incorporated herein by reference.

In the construction of the expression vector, the promoter is preferablypositioned approximately the same distance from the heterologoustranscription start site as it is from the transcription start site inits natural setting. As is known in the art, however, some variation inthis distance can be accommodated without loss of promoter function.

Polyadenylation sequences can also be added to the expression vector inorder to increase the efficiency of mRNA translation. Two distinctsequence elements are required for accurate and efficientpolyadenylation: GU or U rich sequences located downstream from thepolyadenylation site and a highly conserved sequence of six nucleotides,AAUAAA, located 11-30 nucleotides upstream. Termination andpolyadenylation signals that are suitable for some embodiments of theinvention include those derived from SV40.

In addition to the elements already described, the expression vector ofsome embodiments of the invention may typically contain otherspecialized elements intended to increase the level of expression ofcloned nucleic acids or to facilitate the identification of cells thatcarry the recombinant DNA. For example, a number of animal virusescontain DNA sequences that promote the extra chromosomal replication ofthe viral genome in permissive cell types. Plasmids bearing these viralreplicons are replicated episomally as long as the appropriate factorsare provided by genes either carried on the plasmid or with the genomeof the host cell.

The vector may or may not include a eukaryotic replicon. If a eukaryoticreplicon is present, then the vector is amplifiable in eukaryotic cellsusing the appropriate selectable marker. If the vector does not comprisea eukaryotic replicon, no episomal amplification is possible. Instead,the recombinant DNA integrates into the genome of the engineered cell,where the promoter directs expression of the desired nucleic acid.

The expression vector of some embodiments of the invention can furtherinclude additional polynucleotide sequences that allow, for example, thetranslation of several proteins from a single mRNA such as an internalribosome entry site (IRES) and sequences for genomic integration of thepromoter-chimeric polypeptide.

Examples for mammalian expression vectors include, but are not limitedto, pcDNA3, pcDNA3.1(+/−), pGL3, pZeoSV2(+/−), pSecTag2, pDisplay,pEF/myc/cyto, pCMV/myc/cyto, pCR3.1, pSinRep5, DH26S, DHBB, pNMT1,pNMT41, pNMT81, which are available from Invitrogen, pCI which isavailable from Promega, pMbac, pPbac, pBK-RSV and pBK-CMV which areavailable from Strategene, pTRES which is available from Clontech, andtheir derivatives.

Expression vectors containing regulatory elements from eukaryoticviruses such as retroviruses can be also used. SV40 vectors includepSVT7 and pMT2. Vectors derived from bovine papilloma virus includepBV-1MTHA, and vectors derived from Epstein Bar virus include pHEBO, andp2O5. Other exemplary vectors include pMSG, pAV009/A⁺, pMT010/A⁺,pMAMneo-5, baculovirus pDSVE, and any other vector allowing expressionof proteins under the direction of the SV-40 early promoter, SV-40 laterpromoter, metallothionein promoter, murine mammary tumor virus promoter,Rous sarcoma virus promoter, polyhedrin promoter, or other promotersshown effective for expression in eukaryotic cells.

As described above, viruses are very specialized infectious agents thathave evolved, in many cases, to elude host defense mechanisms.Typically, viruses infect and propagate in specific cell types. Thetargeting specificity of viral vectors utilizes its natural specificityto specifically target predetermined cell types and thereby introduce arecombinant gene into the infected cell. Thus, the type of vector usedby some embodiments of the invention will depend on the cell typetransformed. The ability to select suitable vectors according to thecell type transformed is well within the capabilities of the ordinaryskilled artisan and as such no general description of selectionconsideration is provided herein. For example, bone marrow cells can betargeted using the human T cell leukemia virus type I (HTLV-I) andkidney cells may be targeted using the heterologous promoter present inthe baculovirus Autographa californica nucleopolyhedrovirus (AcMNPV) asdescribed in Liang C Y et al., 2004 (Arch Virol. 149: 51-60).

Various methods can be used to introduce the expression vector of someembodiments of the invention into stem cells. Such methods are generallydescribed in Sambrook et al., Molecular Cloning: A Laboratory Manual,Cold Springs Harbor Laboratory, New York (1989, 1992), in Ausubel etal., Current Protocols in Molecular Biology, John Wiley and Sons,Baltimore, Md. (1989), Chang et al., Somatic Gene Therapy, CRC Press,Ann Arbor, Mich. (1995), Vega et al., Gene Targeting, CRC Press, AnnArbor Mich. (1995), Vectors: A Survey of Molecular Cloning Vectors andTheir Uses, Butterworths, Boston Mass. (1988) and Gilboa et al.,[Biotechniques 4 (6): 504-512, 1986] and include, for example, stable ortransient transfection, lipofection, electroporation and infection withrecombinant viral vectors. In addition, see U.S. Pat. Nos. 5,464,764 and5,487,992 for positive-negative selection methods.

Introduction of nucleic acids by viral infection offers severaladvantages over other methods such as lipofection and electroporation,since higher transfection efficiency can be obtained due to theinfectious nature of viruses.

According to one embodiment, the recombinant protein is modified suchthat it is covalently attached to a cell penetrating peptide (CPP).

According to still another embodiment, the cells are stem cells (e.g.mesenchymal stem cells, embryonic stem cells, induced pluripotent stemcells) which have been ex vivo differentiated so as to express(preferably secrete) a factor useful for the treatment of a disease.

Ex vivo differentiated can be effected using methods known in the artincluding by genetic modification and/or culturing in a differentiationmedium that comprises growth factors and other factors known to inducedifferentiation.

Exemplary cells include immune cell, stem cell, progenitor cell, isletcell, bone marrow cells, hematopoietic cells, tumor cells, lymphocytes,leukocytes, granulocytes, hepatocytes, monocytes, macrophages,fibroblasts, neural cells, mesenchymal stem cells, embryonic stem cells,neural stem cells, or other cell with regenerative properties andcombinations thereof.

As mentioned hereinabove, the cells of the present invention can bederived from either autologous sources or from allogeneic sources suchas human cadavers or donors. Since non-autologous cells are likely toinduce an immune reaction when administered to the body severalapproaches have been developed to reduce the likelihood of rejection ofnon-autologous cells. These include either suppressing the recipientimmune system or encapsulating the non-autologous cells inimmunoisolating, semipermeable membranes before transplantation.

Encapsulation techniques are generally classified as microencapsulation,involving small spherical vehicles and macroencapsulation, involvinglarger flat-sheet and hollow-fiber membranes (Uludag, H. et al.Technology of mammalian cell encapsulation. Adv Drug Deliv Rev. 2000;42: 29-64).

Methods of preparing microcapsules are known in the arts and include forexample those disclosed by Lu M Z, et al., Cell encapsulation withalginate and alpha-phenoxycinnamylidene-acetylated poly(allylamine).Biotechnol Bioeng. 2000, 70: 479-83, Chang™ and Prakash S. Proceduresfor microencapsulation of enzymes, cells and genetically engineeredmicroorganisms. Mol Biotechnol. 2001, 17: 249-60, and Lu M Z, et al., Anovel cell encapsulation method using photosensitive poly(allylaminealpha-cyanocinnamylideneacetate). J Microencapsul. 2000, 17: 245-51.

For example, microcapsules are prepared by complexing modified collagenwith a ter-polymer shell of 2-hydroxyethyl methylacrylate (HEMA),methacrylic acid (MAA) and methyl methacrylate (MMA), resulting in acapsule thickness of 2-5 μm. Such microcapsules can be furtherencapsulated with additional 2-5 μm ter-polymer shells in order toimpart a negatively charged smooth surface and to minimize plasmaprotein absorption (Chia, S. M. et al. Multi-layered microcapsules forcell encapsulation Biomaterials. 2002 23: 849-56).

Other microcapsules are based on alginate, a marine polysaccharide(Sambanis, A. Encapsulated islets in diabetes treatment. DiabetesThechnol. Ther. 2003, 5: 665-8) or its derivatives. For example,microcapsules can be prepared by the polyelectrolyte complexationbetween the polyanions sodium alginate and sodium cellulose sulphatewith the polycation poly(methylene-co-guanidine) hydrochloride in thepresence of calcium chloride.

It will be appreciated that cell encapsulation is improved when smallercapsules are used. Thus, the quality control, mechanical stability,diffusion properties, and in vitro activities of encapsulated cellsimproved when the capsule size was reduced from 1 mm to 400 μm (CanapleL. et al, Improving cell encapsulation through size control. J BiomaterSci Polym Ed. 2002; 13:783-96). Moreover, nanoporous biocapsules withwell-controlled pore size as small as 7 nm, tailored surface chemistriesand precise microarchitectures were found to successfully immunoisolatemicroenvironments for cells (Williams D. Small is beautiful:microparticle and nanoparticle technology in medical devices. Med DeviceTechnol. 1999, 10: 6-9; Desai, T. A. Microfabrication technology forpancreatic cell encapsulation. Expert Opin Biol Ther. 2002, 2: 633-46).

Examples of immunosuppressive agents include, but are not limited to,methotrexate, cyclophosphamide, cyclosporine, cyclosporin A,chloroquine, hydroxychloroquine, sulfasalazine (sulphasalazopyrine),gold salts, D-penicillamine, leflunomide, azathioprine, anakinra,infliximab (REMICADE.sup.R), etanercept, TNF.alpha. blockers, abiological agent that targets an inflammatory cytokine, andNon-Steroidal Anti-Inflammatory Drug (NSAIDs). Examples of NSAIDsinclude, but are not limited to acetyl salicylic acid, choline magnesiumsalicylate, diflunisal, magnesium salicylate, salsalate, sodiumsalicylate, diclofenac, etodolac, fenoprofen, flurbiprofen,indomethacin, ketoprofen, ketorolac, meclofenamate, naproxen,nabumetone, phenylbutazone, piroxicam, sulindac, tolmetin,acetaminophen, ibuprofen, Cox-2 inhibitors and tramadol.

The cells of the present invention may be pre-seeded on a scaffold whichis subsequently transplanted into the paranasal sinuses or thesubarachnoid cavity. Alternatively, the scaffold can be pre-transplantedinto the paranasal scaffold or the subarachnoid cavity, following whichthe cells may be transplanted thereto.

As used herein, the term “scaffold” refers to a 3 dimensional matrixupon which cells may be attached and optionally cultured (i.e., surviveand preferably proliferate for a predetermined time period).

Preferably the size of the scaffold is selected such that it does notblock the parasinuses. Contemplated size of a scaffold for insertioninto the paranasal sinus is about 1 mm (thickness)×2-5 cm (diameter).

The scaffold of the present invention may be made uniformly of a singlepolymer, co-polymer or blend thereof. However, it is also possible toform a scaffold according to the invention of a plurality of differentpolymers. There are no particular limitations to the number orarrangement of polymers used in forming the scaffold. Any combinationwhich is biocompatible, may be formed into fibers, and degrades at asuitable rate, may be used. Tissue engineering approaches usinginjectable, in situ gel forming systems to create scaffold inside PNSmay be used.

Both the choice of polymer and the ratio of polymers in a co-polymer maybe adjusted to optimize the stiffness of the scaffold. The molecularweight and cross-link density of the scaffold may also be regulated tocontrol both the mechanical properties of the scaffold and thedegradation rate (for degradable scaffolds). The mechanical propertiesmay also be optimized to mimic those of the tissue at the implant site.The shape and size of the final scaffold should be adapted for theimplant site and tissue type.

Scaffold material may comprise natural or synthetic organic polymersthat can be gelled, or polymerized or solidified (e.g., by aggregation,coagulation, hydrophobic interactions, or cross-linking) into a 3-Dopen-lattice structure that entraps water or other molecules, e.g., toform a hydrogel. Structural scaffold materials may comprise a singlepolymer or a mixture of two or more polymers in a single composition.Additionally, two or more structural scaffold materials may beco-deposited so as to form a polymeric mixture at the site ofdeposition. Polymers used in scaffold material compositions may bebiocompatible, biodegradable and/or bioerodible and may act as adhesivesubstrates for cells. In exemplary embodiments, structural scaffoldmaterials are easy to process into complex shapes and have a rigidityand mechanical strength suitable to maintain the desired shape under invivo conditions.

In certain embodiments, the structural scaffold materials may benon-resorbing or non-biodegradable polymers or materials.

The phrase “non-biodegradable polymer”, as used herein, refers to apolymer or polymers which at least substantially (i.e. more than 50%) donot degrade or erode in vivo. The terms “non-biodegradable” and“non-resorbing” are equivalent and are used interchangeably herein.

Such non-resorbing scaffold materials may be used to fabricate materialswhich are designed for long term or permanent implantation into a hostorganism. In exemplary embodiments, non-biodegradable structuralscaffold materials may be biocompatible. Examples of biocompatiblenon-biodegradable polymers which are useful as scaffold materialsinclude, but are not limited to, polyethylenes, polyvinyl chlorides,polyamides such as nylons, polyesters, rayons, polypropylenes,polyacrylonitriles, acrylics, polyisoprenes, polybutadienes andpolybutadiene-polyisoprene copolymers, neoprenes and nitrile rubbers,polyisobutylenes, olefinic rubbers such as ethylene-propylene rubbers,ethylene-propylene-diene monomer rubbers, and polyurethane elastomers,silicone rubbers, fluoroelastomers and fluorosilicone rubbers,homopolymers and copolymers of vinyl acetates such as ethylene vinylacetate copolymer, homopolymers and copolymers of acrylates such aspolymethylmethacrylate, polyethylmethacrylate, polymethacrylate,ethylene glycol dimethacrylate, ethylene dimethacrylate andhydroxymethyl methacrylate, polyvinylpyrrolidones, polyacrylonitrilebutadienes, polycarbonates, polyamides, fluoropolymers such aspolytetrafluoroethylene and polyvinyl fluoride, polystyrenes,homopolymers and copolymers of styrene acrylonitrile, celluloseacetates, homopolymers and copolymers of acrylonitrile butadienestyrene, polymethylpentenes, polysulfones, polyesters, polyimides,polyisobutylenes, polymethylstyrenes, and other similar compounds knownto those skilled in the art.

In other embodiments, the structural scaffold materials may be a“bioerodible” or “biodegradable” polymer or material.

The phrase “biodegradable polymer” as used herein, refers to a polymeror polymers which degrade in vivo, and wherein erosion of the polymer orpolymers over time occurs concurrent with or subsequent to release ofthe islets. The terms “biodegradable” and “bioerodible” are equivalentand are used interchangeably herein.

Such bioerodible or biodegradable scaffold materials may be used tofabricate temporary structures. In exemplary embodiments, biodegradableor bioerodible structural scaffold materials may be biocompatible.Examples of biocompatible biodegradable polymers which are useful asscaffold materials include, but are not limited to, polylactic acid,polyglycolic acid, polycaprolactone, and copolymers thereof, polyesterssuch as polyglycolides, polyanhydrides, polyacrylates, polyalkylcyanoacrylates such as n-butyl cyanoacrylate and isopropylcyanoacrylate, polyacrylamides, polyorthoesters, polyphosphazenes,polypeptides, polyurethanes, polystyrenes, polystyrene sulfonic acid,polystyrene carboxylic acid, polyalkylene oxides, alginates, agaroses,dextrins, dextrans, polyanhydrides, biopolymers such as collagens andelastin, alginates, chitosans, glycosaminoglycans, and mixtures of suchpolymers. In still other embodiments, a mixture of non-biodegradable andbioerodible and/or biodegradable scaffold materials may be used to forma biomimetic structure of which part is permanent and part is temporary.

PLA, PGA and PLA/PGA copolymers are particularly useful for forming thescaffolds of the present invention. PLA polymers are usually preparedfrom the cyclic esters of lactic acids. Both L(+) and D(−) forms oflactic acid can be used to prepare the PLA polymers, as well as theoptically inactive DL-lactic acid mixture of D(−) and L(+) lactic acids.PGA is the homopolymer of glycolic acid (hydroxyacetic acid). In theconversion of glycolic acid to poly(glycolic acid), glycolic acid isinitially reacted with itself to form the cyclic ester glycolide, whichin the presence of heat and a catalyst is converted to a high molecularweight linear-chain polymer. The erosion of the polyester scaffold isrelated to the molecular weights. The higher molecular weights, weightaverage molecular weights of 90,000 or higher, result in polymerscaffolds which retain their structural integrity for longer periods oftime; while lower molecular weights, weight average molecular weights of30,000 or less, result in both slower release and shorter scaffoldlives. For example, poly(lactide-co-glycolide) (50:50) degrades in aboutsix weeks following implantation.

In an exemplary embodiment, scaffold materials may comprise naturallyoccurring substances, such as, fibrinogen, fibrin, thrombin, chitosan,collagen, alginate, poly(N-isopropylacrylamide), hyaluronate, albumin,collagen, synthetic polyamino acids, prolamines, polysaccharides such asalginate, heparin, and other naturally occurring biodegradable polymersof sugar units.

In certain embodiments, structural scaffold materials may be ionichydrogels, for example, ionic polysaccharides, such as alginates orchitosan. Ionic hydrogels may be produced by cross-linking the anionicsalt of alginic acid, a carbohydrate polymer isolated from seaweed, withions, such as calcium cations. The strength of the hydrogel increaseswith either increasing concentrations of calcium ions or alginate. Forexample, U.S. Pat. No. 4,352,883 describes the ionic cross-linking ofalginate with divalent cations, in water, at room temperature, to form ahydrogel matrix. In general, these polymers are at least partiallysoluble in aqueous solutions, e.g., water, or aqueous alcohol solutionsthat have charged side groups, or a monovalent ionic salt thereof. Thereare many examples of polymers with acidic side groups that can bereacted with cations, e.g., poly(phosphazenes), poly(acrylic acids), andpoly(methacrylic acids). Examples of acidic groups include carboxylicacid groups, sulfonic acid groups, and halogenated (preferablyfluorinated) alcohol groups. Examples of polymers with basic side groupsthat can react with anions are poly(vinyl amines), poly(vinyl pyridine),and poly(vinyl imidazole). Polyphosphazenes are polymers with backbonesconsisting of nitrogen and phosphorous atoms separated by alternatingsingle and double bonds. Each phosphorous atom is covalently bonded totwo side chains. Polyphosphazenes that can be used have a majority ofside chains that are acidic and capable of forming salt bridges with di-or trivalent cations. Examples of acidic side chains are carboxylic acidgroups and sulfonic acid groups. Bioerodible polyphosphazenes have atleast two differing types of side chains, acidic side groups capable offorming salt bridges with multivalent cations, and side groups thathydrolyze under in vivo conditions, e.g., imidazole groups, amino acidesters, glycerol, and glucosyl. Bioerodible or biodegradable polymers,i.e., polymers that dissolve or degrade within a period that isacceptable in the desired application (usually in vivo therapy), willdegrade in less than about five years or in less than about one year,once exposed to a physiological solution of pH 6-8 having a temperatureof between about 25° C. and 38° C. Hydrolysis of the side chain resultsin erosion of the polymer. Examples of hydrolyzing side chains areunsubstituted and substituted imidizoles and amino acid esters in whichthe side chain is bonded to the phosphorous atom through an aminolinkage.

Typically, the scaffolds of the present invention are porous. Theporosity of the scaffold may be controlled by a variety of techniquesknown to those skilled in the art. The minimum pore size and degree ofporosity is dictated by the need to provide enough room for the cellsand for nutrients to filter through the scaffold to the cells. Themaximum pore size and porosity is limited by the ability of the scaffoldto maintain its mechanical stability after seeding. As the porosity isincreased, use of polymers having a higher modulus, addition of stifferpolymers as a co-polymer or mixture, or an increase in the cross-linkdensity of the polymer may all be used to increase the stability of thescaffold with respect to cellular contraction.

The scaffolds may be made by any of a variety of techniques known tothose skilled in the art. Salt-leaching, porogens, solid-liquid phaseseparation (sometimes termed freeze-drying), and phase inversionfabrication may all be used to produce porous scaffolds. Fiber pullingand weaving (see, e.g. Vacanti, et al., (1988) Journal of PediatricSurgery, 23: 3-9) may be used to produce scaffolds having more alignedpolymer threads. Those skilled in the art will recognize that standardpolymer processing techniques may be exploited to create polymerscaffolds having a variety of porosities and microstructures.

Scaffold materials are readily available to one of ordinary skill in theart, usually in the form of a solution (suppliers are, for example, BDH,United Kingdom, and Pronova Biomedical Technology a.s. Norway). For ageneral overview of the selection and preparation of scaffoldingmaterials, see the American National Standards Institute publication No.F2064-00 entitled Standard Guide for Characterization and Testing ofAlginates as Starting Materials Intended for Use in Biomedical andTissue Engineering Medical Products Applications”.

Therapeutic compounds or agents that modify cellular activity can alsobe incorporated (e.g. attached to, coated on, embedded or impregnated)into the scaffold material. Campbell et al (US Patent Application No.20030125410) which is incorporated by reference as if fully set forth byreference herein, discloses methods for fabrication of 3D scaffolds forstem cell growth, the scaffolds having preformed gradients oftherapeutic compounds. The scaffold materials, according to Campbell etal, fall within the category of “bio-inks”. Such “bio-inks” are suitablefor use with the compositions and methods of the present invention.

Exemplary agents that may be incorporated into the scaffold of thepresent invention include, but are not limited to those that promotecell adhesion (e.g. fibronectin, integrins), cell colonization, cellproliferation, cell differentiation, cell extravasation and/or cellmigration. Thus, for example, the agent may be an amino acid, a smallmolecule chemical, a peptide, a polypeptide, a protein, a DNA, an RNA, alipid and/or a proteoglycan.

Proteins that may be incorporated into the scaffolds of the presentinvention include, but are not limited to extracellular matrix proteins,cell adhesion proteins, growth factors, cytokines, hormones, proteasesand protease substrates. Thus, exemplary proteins include vascularendothelial-derived growth factor (VEGF), activin-A, retinoic acid,epidermal growth factor, bone morphogenetic protein, TGFβ, hepatocytegrowth factor, platelet-derived growth factor, TGFα, IGF-I and II,hematopoetic growth factors, heparin binding growth factor, peptidegrowth factors, erythropoietin, interleukins, tumor necrosis factors,interferons, colony stimulating factors, basic and acidic fibroblastgrowth factors, nerve growth factor (NGF) or muscle morphogenic factor(MMP). The particular growth factor employed should be appropriate tothe desired cell activity. The regulatory effects of a large family ofgrowth factors are well known to those skilled in the art.

Since it has been observed that the initial distribution of cells withinthe scaffold after seeding is related to the cell densities subsequentlyachieved, methods of cell seeding require careful consideration. Thus,cells can be seeded in a scaffold by static loading, or, morepreferably, by seeding in stirred flask bioreactors (scaffold istypically suspended from a solid support), in a rotating wall vessel, orusing direct perfusion of the cells in medium in a bioreactor. Highestcell density throughout the scaffold is achieved by the latter (directperfusion) technique.

The cells may be seeded directly onto the scaffold, or alternatively,the cells may be mixed with a gel which is then absorbed onto theinterior and exterior surfaces of the scaffold and which may fill someof the pores of the scaffold. Capillary forces will retain the gel onthe scaffold before hardening, or the gel may be allowed to harden onthe scaffold to become more self-supporting. Alternatively, the cellsmay be combined with a cell support substrate in the form of a geloptionally including extracellular matrix components. An exemplary gelis Matrigel™, from Becton-Dickinson. Matrigel™ is a solubilized basementmembrane matrix extracted from the EHS mouse tumor (Kleinman, H. K., etal., Biochem. 25:312, 1986). The primary components of the matrix arelaminin, collagen I, entactin, and heparan sulfate proteoglycan(perlecan) (Vukicevic, S., et al., Exp. Cell Res. 202:1, 1992).Matrigel™ also contains growth factors, matrix metalloproteinases (MMPs[collagenases]), and other proteinases (plasminogen activators [PAs])(Mackay, A. R., et al., BioTechniques 15:1048, 1993). The matrix alsoincludes several undefined compounds (Kleinman, H. K., et al., Biochem.25:312, 1986; McGuire, P. G. and Seeds, N. W., J. Cell. Biochem. 40:215,1989), but it does not contain any detectable levels of tissueinhibitors of metalloproteinases (TIMPs) (Mackay, A. R., et al.,BioTechniques 15:1048, 1993). Alternatively, the gel may begrowth-factor reduced Matrigel, produced by removing most of the growthfactors from the gel (see Taub, et al., Proc. Natl. Acad. Sci. USA(1990); 87 (10:4002-6). In another embodiment, the gel may be a collagenI gel, alginate, or agar. Such a gel may also include otherextracellular matrix components, such as glycosaminoglycans, fibrin,fibronectin, proteoglycans, and glycoproteins. The gel may also includebasement membrane components such as collagen IV and laminin Enzymessuch as proteinases and collagenases may be added to the gel, as maycell response modifiers such as growth factors and chemotactic agents.

The cells of the present invention may be transplanted per se, or aspart of a pharmaceutical composition.

As used herein a “pharmaceutical composition” refers to a preparation ofone or more of the cell populations described herein with other chemicalcomponents such as physiologically suitable carriers and excipients. Thepurpose of the pharmaceutical composition is to facilitateadministration of the active ingredients to the subject.

Herein the term “active ingredient” refers to the agents, which increasethe amount or activity of the lysosomal enzymes in the brain.

Hereinafter, the phrases “physiologically acceptable carrier” and“pharmaceutically acceptable carrier” which may be interchangeably usedrefer to a carrier or a diluent that does not cause significantirritation to the subject and does not abrogate the biological activityand properties of the administered active ingredients. An adjuvant isincluded under these phrases.

Herein, the term “excipient” refers to an inert substance added to thepharmaceutical composition to further facilitate administration of anactive ingredient of the present invention. Examples, withoutlimitation, of excipients include calcium carbonate, calcium phosphate,various sugars and types of starch, cellulose derivatives, gelatin,vegetable oils and polyethylene glycols.

Techniques for formulation and administration of drugs may be found in“Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa.,latest edition, which is incorporated herein by reference.

The pharmaceutical composition may be manufactured by processes wellknown in the art, e.g., by means of conventional mixing, dissolving,granulating, dragee-making, levigating, emulsifying, encapsulating,entrapping or lyophilizing processes.

The pharmaceutical composition may comprise a proteolytic enzymeinhibitor so as to prevent the hydrolysis of the desired peptide andprotein drug (e.g. insulin) in the nasal cavity and, thus, improve thestability of the protein drug at the absorption site. For example,camostatmesilate, an aminopeptidase and trypsin inhibitor, improved thenasal delivery of vasopressin and desmopressin.

Additionally, or alternatively, the pharmaceutical composition maycomprise an absorption enhancer. Preferably, the absorption promotershould be rapid-acting, resulting in transient and reversible modulationof the absorptive properties or physiology of the nasal mucosa, and notbe absorbed systemically. Further, it should be devoid of any toxic,irritating or allergic activity. The degree of absorption enhancementshould be predictable and reproducible. They should also not permitentry of potentially dangerous environmental materials and should becompatible with drugs and adjuvants in the preparation. Examples ofcompounds that may be used as absorption enhances include saponin,sodium deoxycholate, ethylendiamine tetra-Acetic Acid (EDTA) andlecithin, surfactants, cationic polymers, chitosan and its derivatives,poly-L-arginine, cationized gelatin, cyclodextrin and its derivatives,tight junction modulating lipids, tight junction modulating peptides,nitric oxide donors, N-acetyl-L-cysteine, bile salt and its derivatives,and fatty acid and its derivatives and others from the below list.

1% sodium deoxycholate, 1% sodium taurodihydrofusidate (STDHF), 0.5%lysophosphatidylcholine (LPC), 0.125% dodecylmaltoside, 0.5% Laureth-9,0.5% sucrose cocoate, 3.5% soybean-derived sterol, 1.0% sterolglucoside, Cyclodextrins (5% DM-bCD, 5% DM-bCD, 30% DM-bCD 2, DM-bCD, 5%DM-bCD, 5% α-CD); Cell-penetrating peptides (0.5 mM L-R8, 0.5 mM D-R8,0.5 mM D-penetratin, 0.5 mM L-penetratin, 0.5 mM shuffle (R,K fix));Cationized polymers (0.5% chitosan, 85.7% chitosan, 0.2% sperminatedgelatin, 0.2% aminated H-gelatin, 0.4% aminated H-gelatin, 0.2% aminatedH-gelatin, 0.2% aminated L-gelatin) and Chelators (e.g. 0.5% EDTA-2Na).

Further information on absorption enhancers may be found in Duan et al.,Drug Discovery Today, Volume 15, Numbers 11/12, June 2010, incorporatedherein by reference.

The present invention further contemplates using different methods toachieve mucoadhesion, thereby enhancing bioavailability.

Bioadhesive microsphere delivery system: To avoid rapid clearance owingto ciliary beating and prolong the residence time in the nasal mucosa,the present invention contemplates the use of crosslinked dextranmicrosphere, starch microsphere, aminated gelatin microspheres orhyaluronic acid ester microspheres. Other types of bioadhesivemicrosphere delivery systems such as biadhesive powders are disclosed inDuan et al., Drug Discovery Today, Volume 15, Numbers 11/12, June 2010,incorporated herein by reference.

The pharmaceutical composition should contain the active ingredients inan amount effective to achieve disease treatment.

Determination of a therapeutically effective amount is well within thecapability of those skilled in the art, especially in light of thedetailed disclosure provided herein.

For any preparation used in the methods of the invention, thetherapeutically effective amount or dose can be estimated initially fromin vitro and cell culture assays. Following this, a dose can beformulated in animal models (e.g. streptozotocin STZ rats) to achieve adesired plasma concentration or titer. To find a minimal dose ofpancreatic islets required for diabetes reversal, aliquots of 500, 1000,1500 and 2000 islets may initially be implanted in diabetic rats. Suchinformation can be used to more accurately determine useful doses inhumans.

Toxicity and therapeutic efficacy of the active ingredients describedherein can be determined by standard pharmaceutical procedures in vitro,in cell cultures or experimental animals. The data obtained from thesein vitro and cell culture assays and animal studies can be used informulating a range of dosage for use in human. The dosage may varydepending upon the dosage form employed and the route of administrationutilized. The exact formulation, route of administration and dosage canbe chosen by the individual physician in view of the patient'scondition. (See e.g., Fingl, et al., 1975, in “The Pharmacological Basisof Therapeutics”, Ch. 1 p. 1).

Dosage amount and interval may be adjusted individually to provideplasma or brain levels of the active ingredients which are sufficient toachieve the desired therapeutic effect (minimal effective concentration,MEC). The MEC will vary for each preparation, but can be estimated fromin vitro data. Dosages necessary to achieve the MEC will depend onindividual characteristics and route of administration. Detection assayscan be used to determine plasma concentrations.

Depending on the severity and responsiveness of the condition to betreated, dosing can be of a single or a plurality of administrations,with course of treatment lasting from several days to several weeks oruntil cure is effected or diminution of the disease state is achieved.

The amount of the composition to be administered will be dependent onthe subject being treated, the severity of the affliction, the manner ofadministration, the judgment of the prescribing physician, etc.

Compositions of the present invention may, if desired, be presented in apack or dispenser device, such as an FDA approved kit, which may containone or more unit dosage forms containing the active ingredients. Thepack may, for example, comprise metal or plastic foil, such as a blisterpack. The pack or dispenser device may be accompanied by instructionsfor administration. The pack or dispenser may also be accommodated by anotice associated with the container in a form prescribed by agovernmental agency regulating the manufacture, use or sale ofpharmaceuticals, which notice is reflective of approval by the agency ofthe form of the compositions or human or veterinary administration. Suchnotice, for example, may be of labeling approved by the U.S. Food andDrug Administration for prescription drugs or of an approved productinsert.

Transplantation of the cells into the nose, and more specifically to theparanasal sinuses (e.g. the maxillary sinus) may be effected in anon-invasive manner by inserting a flexible (e.g. malleable) tube whichenters the ostium which is localized under the median nasal concha.Preferably, when inserted into the nose, the end of the tube reaches theparanasal sinus cavity, prior to allowing release of the cells.

Thus, according to another aspect of the present invention there isprovided an endoscopic device comprising a container attached to aflexible tubing wherein the inner surface of the container is coatedwith an agent that prevents adhesiveness of cell aggregates thereto.

The device generally includes a tubing, e.g., a catheter, attached to acontainer having an elongate pusher at its end. The tubing may beflexible or rigid, or may be designed to have varying degrees ofstiffness along its length, e.g., the distal portion of the tubing maybe stiffer than the proximal portion. In addition, the distal portion ofthe tubing may be variously angulated to facilitate positioning andadvancement of the conduit through the sinus ostium. For example, thedistal portion may be angulated from about 0 degree to about 175 degree,from about 0 degree to about 135 degree, or from about 0 degree to about90 degree.

The diameter of the lumen of the tubing is typically between about200-1000 μm or 100-500 μm and the tubing is typically about 5-20 cm inlength. The tubing may be made from any biocompatible materialincluding, but not limited to, stainless steel and any of its alloys;titanium alloys, e.g., nickel-titanium alloys; polymers, e.g.,polyethylene and copolymers thereof, polyethylene terephthalate orcopolymers thereof, nylon, silicone, polyurethanes, fluoropolymers,poly(vinylchloride), and combinations thereof, depending on the amountof flexibility or stiffness desired. The pusher and container may bemade from similar materials.

The tubing is attached to a container for holding the cells. Thecontainer is coated on its inner surface with an agent that preventscell aggregates from sticking thereto. The surface of the pusher whichcontacts the cells may be coated with a similar agent. Such agentsinclude, but are not limited to silicon and polyethylene. Once accessthrough a sinus ostium has been obtained with the tubing, the pusherslidably engages the cells and is advanced until the cells exit thecontainer and advance through the tubing into the sinus. An optic fibermay also be used while positioning the tubing to aid with visualizationof the ostium. The optic fiber may be incorporated into the device ofthe present invention or may be used separately (e.g. by using anendoscope).

In certain cases, e.g., when ostia are closed or difficult to access,cell transplantation into one or more sinuses may be completed throughthe sinus wall using a sharp-tipped conduit, e.g., a needle, trocar, orangiocatheter, with or without visualization using computer image-guidedtechnology or endoscopy. Once the appropriate access point for the sinushas been determined, force is applied to the sharp-tipped conduit sothat it punctures the sinus wall. Advancement of a pusher throughcontainer then deposits the cells into the sinus.

FIG. 4 shows an exemplary transplantation device 10. The device includesa population of cells 12, a tubing 14 having a lumen 16, wherein thetubing 14 is attached to a container 18 into which is inserted a pusher20. The pusher 20 may further comprise a handle 24 for easy grasp. Theinside walls of the container 18 are coated with an agent which preventssticking of cellular material. The device may further incorporate anoptic fiber 22 for visualization. The pusher 20 is advanced distallywithin the container 18 to slidably engage the cells 20 and move it upthe container 18 through the tubing 14 into the sinus. Although the tipof the tubing is shown to be blunt in FIG. 4, it may also be sharpand/or beveled, usually depending on the implant delivery method.

A force applied to the pusher 20 allows a predetermined amount of cells12 into the tubing 14, e.g., by contact with a pusher or pressurizedgas, could be used to deliver the cells 12 into the sinus.

As mentioned, the device described herein may incorporate, or may beused in conjunction with, endoscopes. Such endoscopes will typicallyinclude light transmitting optical fibers for casting light in the areato be viewed by the scope and image transmitting optical fibers forcarrying an image received by the scope to an eyepiece or monitor devicelocated outside the patient's body. In some embodiments a scope, such asa disposable and/or flexible scope, may be affixed to the workingdevice. Examples of such endoscopes that are suitable for incorporationinto the working devices of this invention include that described inU.S. Pat. Nos. 4,708,434; 4,919,112; 5,127,393; 5,519,532; 5,171,233,5,549,542, 6,551,239 and 6,572,538 as well as published United StatesPatent Application No. 2001/0029317A1, the entireties of which areexpressly incorporated herein by reference.

Alternatively, transplantation into the paranasal sinuses may beeffected using a minimally invasive method whereby a needle is used toenter the sinus via a thin bone under the lower concha.

A more invasive means of carrying out the transplantation is byperforming a small cut in the upper gum of the mouth and introducing aneedle through the bone into the paranasal sinus. Another example is touse an opening in the upper tooth for cell transplantation into themaxillary sinus.

Transplantation into the subarachnoid cavity may be effected by methodsand devices for cell delivery in CNS such as described by Potts M B,Silvestrini M T, Lim D A. Devices for cell transplantation into thecentral nervous system: Design considerations and emerging technologies.Surg Neurol Int. 2013 Mar. 19; 4 (Suppl 1):S22-30. doi:10.4103/2152-7806.109190).

As used herein the term “about” refers to ±10%.

The terms “comprises”, “comprising”, “includes”, “including”, “having”and their conjugates mean “including but not limited to”.

The term “consisting of” means “including and limited to”.

The term “consisting essentially of” means that the composition, methodor structure may include additional ingredients, steps and/or parts, butonly if the additional ingredients, steps and/or parts do not materiallyalter the basic and novel characteristics of the claimed composition,method or structure.

Throughout this application, various embodiments of this invention maybe presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 3, 4, 5, and 6. This appliesregardless of the breadth of the range.

As used herein the term “method” refers to manners, means, techniquesand procedures for accomplishing a given task including, but not limitedto, those manners, means, techniques and procedures either known to, orreadily developed from known manners, means, techniques and proceduresby practitioners of the chemical, pharmacological, biological,biochemical and medical arts.

As used herein, the term “treating” includes abrogating, substantiallyinhibiting, slowing or reversing the progression of a condition,substantially ameliorating clinical or aesthetical symptoms of acondition or substantially preventing the appearance of clinical oraesthetical symptoms of a condition.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable subcombination or as suitable in any other describedembodiment of the invention. Certain features described in the contextof various embodiments are not to be considered essential features ofthose embodiments, unless the embodiment is inoperative without thoseelements.

Various embodiments and aspects of the present invention as delineatedhereinabove and as claimed in the claims section below find experimentalsupport in the following examples.

EXAMPLES

Reference is now made to the following examples, which together with theabove descriptions illustrate some embodiments of the invention in a nonlimiting fashion.

Generally, the nomenclature used herein and the laboratory proceduresutilized in the present invention include molecular, biochemical,microbiological and recombinant DNA techniques. Such techniques arethoroughly explained in the literature. See, for example, “MolecularCloning: A laboratory Manual” Sambrook et al., (1989); “CurrentProtocols in Molecular Biology” Volumes I-III Ausubel, R. M., ed.(1994); Ausubel et al., “Current Protocols in Molecular Biology”, JohnWiley and Sons, Baltimore, Md. (1989); Perbal, “A Practical Guide toMolecular Cloning”, John Wiley & Sons, New York (1988); Watson et al.,“Recombinant DNA”, Scientific American Books, New York; Birren et al.(eds) “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, ColdSpring Harbor Laboratory Press, New York (1998); methodologies as setforth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis,J. E., ed. (1994); “Culture of Animal Cells—A Manual of Basic Technique”by Freshney, Wiley-Liss, N. Y. (1994), Third Edition; “Current Protocolsin Immunology” Volumes I-III Coligan J. E., ed. (1994); Stites et al.(eds), “Basic and Clinical Immunology” (8th Edition), Appleton & Lange,Norwalk, Conn. (1994); Mishell and Shiigi (eds), “Selected Methods inCellular Immunology”, W. H. Freeman and Co., New York (1980); availableimmunoassays are extensively described in the patent and scientificliterature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153;3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654;3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219;5,011,771 and 5,281,521; “Oligonucleotide Synthesis” Gait, M. J., ed.(1984); “Nucleic Acid Hybridization” Hames, B. D., and Higgins S. J.,eds. (1985); “Transcription and Translation” Hames, B. D., and HigginsS. J., eds. (1984); “Animal Cell Culture” Freshney, R. I., ed. (1986);“Immobilized Cells and Enzymes” IRL Press, (1986); “A Practical Guide toMolecular Cloning” Perbal, B., (1984) and “Methods in Enzymology” Vol.1-317, Academic Press; “PCR Protocols: A Guide To Methods AndApplications”, Academic Press, San Diego, Calif. (1990); Marshak et al.,“Strategies for Protein Purification and Characterization—A LaboratoryCourse Manual” CSHL Press (1996); all of which are incorporated byreference as if fully set forth herein. Other general references areprovided throughout this document. The procedures therein are believedto be well known in the art and are provided for the convenience of thereader. All the information contained therein is incorporated herein byreference.

Example 1 Transplantation of Insulin-Producing Beta Cell Line (Ins-1)into PNS of Diabetic Rats

Materials and Methods

Inbred Lewis rats (males, 270-280 g) were used as recipient forinsulinoma cell (INS-1) transplantation. Insulin dependent diabetes wasinduced by iv injection of streptozotocin (STZ) at 85 mg/kg body weight.This dose of STZ results in a stable and spontaneously irreversiblediabetes in Lewis rats. Rats were pretreated with 10 mg/kg cyclosporineA (CyA) 2 days before transplantation followed by a daily dosage of CyA.One week after STZ injection, about 30×10⁶ INS-1 cells were transplanted(Tx) into the PNS of diabetic rats.

Due to the relatively small rat PNS volume, noninvasive cell deliveryinto the rat PNS is complicated. (The total volume of rat PNS is ˜50 mm3compared to a very large human PNS occupying ˜60-80 cm3 space). Thus, asimple surgical procedure was used and recipient animals wereanesthetized with ketamine/xylozine, followed by a 0.5-cm longitudinalincision of skin between the eyes. The plate of the bone was thenexposed and trepanized with a 1 mm burr in the direction of the PNS. Analiquot of cells (˜5 μl) was introduced into the PNS using a pipettetip.

Head and pancreas were removed for immunohistochemical analysis twoweeks after cell implantation as described previously (Bloch K, et al.,Acta Biomater. 2010; 6(3):1200-5; Bloch K, et al., Histochem Cell Biol.2012; 137(6):801-10).

Results

Reverse of diabetes was shown after transplantation of insulin-producingbeta cell line (INS-1) into PNS of STZ-diabetic Lewis rats treated withcyclosporine A (CyA) (FIG. 2) Immunohistochemical analysis indicatesinsulin staining of INS-1 cells (brown color) localized in PNS aftertwo-week post-transplantation period (FIGS. 3A-B) Immunostaining ofblood vessel endothelial cells with vWF shows intensiveneovascularization of grafted tissue (Data not shown).

Example 2 Islet Transplantation in a Subarachnoid Cavity SurroundingOlfactory Bulb of Diabetic Rats

In this example, the present inventors studied the different parametersof glucose homeostasis and islet morphology following islettransplantation in a subarachnoid cavity of diabetic rats. Thistransplantation procedure enabled the assembly of grafted isletsdirectly onto the glomeruli of the olfactory bulb.

Materials and Methods

Chemicals:

The CMRL and RPMI 1640 culture media, Hanks balanced saline solution(HBSS), fetal calf serum (FCS), penicillin, streptomycin, and otherreagents for tissue culture were obtained from Biological Industries(Beit Haemek, Israel); Collagenase NB8 from Serva (Heidelberg, Germany);Bovine serum albumin (BSA), bovine DNAse, streptozotocin (STZ) andhistopaque were acquired from Sigma-Aldrich (St Louis, Mo., USA);Dithizone (DTZ) was obtained from Merck (Darmstadt, Germany). RatC-peptide ELISA kit was purchased from Mercodia AB (Uppsala, Sweden),the primary guinea pig anti-insulin from Progen GmbH (Heidelberg,Germany), mouse anti-glucagon antibodies from Sigma (St Louis, Mo.,USA), Antibody diluent and fluorescent mounting medium were purchasedfrom Dako (Carpinteria, Calif., USA). Secondary goat anti-rabbit,anti-mouse and anti-guinea pig antibodies (Cy^(tm2) and Cy^(tm3)) andperoxidase-conjugated goat anti-rabbit Ig were purchased from JacksonImmuno Research Laboratories Inc. (Baltimore, Pa., USA). Adhesionmicroscopic slides were from Marieneld GmbH & Co. KG-(Lauda-Konigshofen,Germany).

Animals and Diabetes Induction:

10-12-week-old male Lewis inbred rats (Harlan, Jerusalem, Israel)weighing 260-280 g, were used in all experiments. All procedures wereapproved by the Institutional Animal Care and Use Committee. Diabeteswas induced by a single intravenous injection of 85 mg STZ/kg bodyweight. STZ was injected after being dissolved in citrate buffer (pH4.5). Only animals with persistent hyperglycemia (>350 mg/dl) were usedas transplant recipients.

Pancreatic Islet Isolation and Dithizone (DTZ) Staining:

Islets were isolated from pancreata of Lewis rats by enzymatic digestionwith collagenase solution containing 12 PZ units/ml collagenase NB8 and1 mg/mL bovine DNAse in HBSS solution for 15 min at 37° C. Islets werepurified using a discontinuous Histopaque gradient density and collectedfrom the 1.077 interphase. The islets were then washed and cultured in 8ml CMRL:RPMI medium 1640 (1:1) supplemented with 10% FCS and 1%antibiotics, overnight in a CO₂ incubator (5% CO₂ /95% air) before beingtransplanted. Islet quantity was expressed as the number of IEQ, whichis calculated based on the number and diameter of the DTZ-stained isletsin four aliquots of 150 ul from islet suspension using the standard IEQconversion factors. The number of IEQ obtained per pancreas varied from500 to 600.

The morphology of isolated islets was analyzed after DTZ-staining usingan inverted microscope. DTZ stock solution was prepared by dissolving 50mg of DTZ in 5 ml of dimethyl sulfoxide. For in vitro staining, 10 μl ofDTZ-stock solution was dissolved in 1 ml of phenol red free RPMI medium1640. Culture medium was replaced by DTZ solution. The stainingprocedure was performed at 37° C. for 15 minutes in a CO₂-incubator.Islets stained with DTZ were washed twice with fresh phenol red freeRPMI medium 1640, analyzed by an inverted microscope and images werecaptured.

Islet Transplantation Procedure:

Animals were anesthetized using ketamine and xylazine (100 mg and 15 mgper kg body weight, respectively). Throughout surgery, the eyes werecovered with ointment to prevent drying out and infection of the cornea.After head fur shaving and disinfection, the skin overlying the skullwas incised using a scalpel, the soft tissue and muscles were removedfrom the surface of the frontal skull. Using a stainless steel burrdisinfected in 70% ethanol, a hole of 1 mm diameter was then made in thefrontal bone 2 mm lateral to the midline at the location shown in FIG.7A. The dura mater and the arachnoid membrane were then perforated and apolyethylene cannula containing 3000 IEQ resuspended in 20 μl of HBSSwas inserted into the subarachnoid space. Following islet introductioninto the subarachnoid cavity by air pressure, the hole was sealed usingbone wax and the incision was closed with surgical suture. Histologicalanalysis revealed localization of grafted islets directly onto theglomeruli of the olfactory bulb (FIG. 5C and FIG. 7B).

Metabolic Follow-Up:

Non-fasting blood glucose concentration was measured between 9.00 am and10.00 am in whole blood with a portable glucometer (Acu-Check; HoffmannLa Roche, Basel Switzerland). An intraperitoneal test tolerance toglucose (IPTTG) was performed in all experimental groups. For IPTTG, 25%dextrose solution was injected intraperitoneally following 6 h fast at adose of 4 μl/g body weight. Blood glucose was measured before and at 15,30, 60 and 120 min post-glucose injection. Body weight was measuredweekly before and after STZ injection. C-peptide level was tested inblood serum using an ELISA kit for rat C-peptide, according tomanufacturer protocol (Mercodia AB, Sweden).

Histological Evaluation of Transplanted Islets and Pancreas:

For histological analysis, the recipients were sacrificed 2 monthsfollowing transplantation. Pancreata and heads were fixed in 10%buffered formalin and processed routinely for histology. Heads wereplaced in Calci-clear rapid (Life Science Products, Inc. Frederick,Colo., USA) to decalcify the bones before embedding in paraffin.Histological sections (4 μm thick) were stained with H&EImmunohistochemical analysis of islet hormone expression andvascularization was performed as described previously [Bloch K, et al.,Histochem Cell Biol 2012; 137: 801; Bloch K, et al., Acta Biomater 2010;6: 1200). Briefly, each section was blocked with 20% normal serum andincubated with primary polyclonal guinea pig anti-insulin antibodies(1:1000) and monoclonal mouse anti-glucagon antibodies (1:2000)overnight, at 4° C. After washing, sections were incubated withsecondary antibodies: peroxidase-conjugated goat anti-guinea pig Ig(1:400) Cy^(tm3)-conjugated affinipure anti-guinea pig Ig (1:400) andanti-mouse Cy^(tm2) (1:400) for one hour at room temperature. Forhorseradish peroxidase (HRP) staining, 3% H₂O₂ was used to blockendogenous peroxidase activity. All dilutions were made with Dakoantibody diluent.

Immunochemistry Controls and Analysis:

Immunostaining of insulin and glucagon was negative when the primaryantibodies were replaced with Dako antibody diluent or normal serum. Thecells were analyzed by light and fluorescent microscopy (BX-52, OlympusLtd, Japan); images were captured by digital camera and merged usingImage-Pro plus v. 5.1.

Statistical Analysis:

Data are expressed as mean±SD of repeated experiments. Student's t test(two-tailed) was used to evaluate the statistical significance ofdifferences between groups.

Results

As shown in FIG. 5A, all severely diabetic rats transplanted with 3000islet equivalents achieved normoglycemia within the first two days whichwas maintained for two months post transplantation. One month and twomonths after transplantation the rats demonstrated normal glucosetolerance typical for healthy animals (FIG. 5B). These data correlatewell with the normalization of blood C-peptide level found intransplanted animals (526±90, 56±47 and 372±129 μmol/L in intact,diabetic and transplanted rats, respectively). In contrast, thenon-transplanted diabetic animals were permanently hyperglycemic,showing impaired glucose tolerance, decreased level of blood C-peptideand body weight loss. They died within the first month after diabetesinduction.

Two months following transplantation, rat heads were fixated informalin, decalcified and processed routinely for histology. Hematoxylinand eosin staining demonstrated the intact morphology of transplantedpancreatic islets located in the subarachnoid cavity between the frontalbone of skull and the olfactory bulb. Microscopic examination ofhistological sections stained for insulin revealed the localization ofislet grafts directly on the glomerular layer of the olfactory bulb(FIG. 5C). In contrast to the intact pancreas, where individual isletsare separated one from another by multilayer exocrine tissue, the isletstransplanted on the surface of the olfactory bulb formed an islet-liketissue, where individual islets joined one another. In order to compareislet cellular architecture in the intact pancreas and followingtransplantation in a subarachnoid cavity, double staining for insulinand glucagon was performed. The arrangement of beta and alpha cells inthe grafted islets was typical for rodents: the insulin producing betacells were found in the central area of islets, while glucagon producingalpha cells had a mantle position (FIG. 5D). Thus, the islettransplantation directly onto the glomeruli of the olfactory bulbresulted in a preservation of islet architecture and a quick reversal ofhyperglycemia to stable normoglycemia.

Additional advantages of this transplantation procedure may be possibleeffects of transplanted islets on improvement of cognitive function. Thecentral nervous system is known to be very sensitive to disruptedinsulin signaling and glucose homeostasis which occur in diabeticpatients. In this regard, we suggest that transplantation of pancreaticislets in the subarachnoid cavity surrounding the olfactory bulb can notonly serve to reverse diabetes, but also to provide adequate insulinsupply to brain, restore cerebral glucose homeostasis and cognitivefunctions. A schematic presentation of possible anti-diabetic andanti-dementia effects of islets transplanted in subarachnoid cavity isshown in FIG. 6.

In conclusion, the achievement of long-term islet graft function insubarachnoid cavity surrounding the olfactory bulb of diabetic ratsprovides an opportunity to clarify the effects of insulin and otherislet hormones secreted inside blood brain barrier on diabetes and toestimate the impact of transplanted islets on various neurodegenerativeand neuropsychological disorders.

Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims.

All publications, patents and patent applications mentioned in thisspecification are herein incorporated in their entirety by referenceinto the specification, to the same extent as if each individualpublication, patent or patent application was specifically andindividually indicated to be incorporated herein by reference. Inaddition, citation or identification of any reference in thisapplication shall not be construed as an admission that such referenceis available as prior art to the present invention. To the extent thatsection headings are used, they should not be construed as necessarilylimiting.

What is claimed is:
 1. A method of treating a neurodegenerative disorderin a subject in need thereof, comprising administering a therapeuticallyeffective amount of insulin secreting cells into the nasal cavity of thesubject or into the subarachnoid cavity, thereby treating the subject.2. The method of claim 1, wherein said cells are pancreatic beta cells.3. The method of claim 1, wherein said cells are cell aggregates.
 4. Themethod of claim 3, wherein said aggregates are greater than 50 micronsin diameter.
 5. The method of claim 1, wherein said cells comprisegenetically modified cells.
 6. The method of claim 1, wherein said cellsare seeded on a scaffold prior to the administering.
 7. The method ofclaim 1, further comprising inserting a scaffold into the nasal cavityor the subarachnoid cavity prior to the administering.
 8. The method ofclaim 1, wherein said cells comprise immunoisolated cells.
 9. The methodof claim 1, wherein said cells are autologous to said subject.
 10. Themethod of claim 1, wherein said cells are non-autologous to saidsubject.
 11. The method of claim 1, wherein said nasal cavity comprisesa paranasal sinus.
 12. The method of claim 11, wherein said paranasalsinus is selected from the group consisting of the maxillary sinuses,frontal sinuses, ethmoid sinuses, and the sphenoid sinuses.
 13. Themethod of claim 1, wherein said administering is effected by endoscopy.14. The method of claim 1, wherein said administering into saidsubarachnoid cavity is performed between the frontal bone of the skulland the olfactory bulb of the subject.
 15. The method of claim 1,wherein said administering into said subarachnoid cavity is performedsuch that at least 20% of said cells localize to the olfactory bulb ofthe subject.
 16. The method of claim 1, wherein said cells are livercells.